Compositions, methods and apparatuses for preserving platelets

ABSTRACT

A platelet composition suitable for direct transfusion into a patient is provided. The platelet composition includes a preservation medium comprising plasma and a gel-forming material in a concentration relative to the plasma such that the medium is in a sufficiently fluent state at about 37° C. to allow platelets to move within the medium and is in a sufficiently gelatinous state at about 5° C. to substantially prevent platelets from moving freely within the medium; and platelets.

CROSS-REFERENCE

This application is a Continuation application of U.S. application Ser.No. 09/267,891, filed Mar. 11, 1999 now U.S. Pat. No. 6,828,090 entitled“COMPOSITIONS, METHODS AND APPARATUSES FOR PRESERVING PLATELETS”, whichis a continuation-in-part of U.S. application Ser. No. 09/183,581, filedOct. 30, 1998 now U.S. Pat. No. 6,413,713 entitled “METHOD AND APPARATUSFOR PRESERVING BIOLOGICAL MATERIALS”, which are both incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to compositions, methods and apparatusesfor preserving biological materials. More particularly, the inventionrelates to compositions, methods and apparatuses for the extendedstorage of platelets.

BACKGROUND OF THE INVENTION

Over the last 40 years the need for the therapeutic use of biologicalmaterials, such as blood, skin and other tissues, kidneys, hearts,livers and other body organs has increased dramatically. Blood andplasmas components, including red cells, platelets, clotting factors,albumin, and antibodies are isolated and used to treat various bleedingproblems. In particular, platelets, essential components of the humanblood, are used extensively for assisting in the control of bleeding andreplacing functionally defective platelets in patients. For example,platelet transfusions are required by trauma patients who have lostsignificant amount of blood, patients undergoing chemotherapy thatreduces the number of platelets and causes functional defects inremaining platelets, and patients with certain platelet-depletingdiseases.

Constituents in whole blood include leukocytes (white blood cells),erythrocytes (red blood cells), thrombocytes, platelets and plasma.Platelets are not entire cells but small detached cell fragments or“minicells” derived from the cortical cytoplasm of large cells calledmegakaryocytes in the bone marrow. Platelets comprise an outer membraneand cytoplasm from the megakaryocytes which contain granules, densebodies, dense tubular system and mitochondria. Platelets adherespecifically to the endothelial cell lining of damaged blood vessels,where they trigger and participate in hemostasis, or clotting, andrelease inflammatory mediators in response to contact with theendothelial cell lining. Important mediators released by plateletsinclude serotonin and coagulation factors. Vascular breaches arerepaired by platelets through adhesion and the response to damage isamplified by platelet secretions resulting in platelet aggregation andfibrin formation, i.e. stabilized clot.

It is very important to preserve platelets after their isolation fromthe body under conditions not only maintaining the biological activityof platelets but also suitable for clinical use. The average survivaltime for a platelet in the body after it leaves the bone marrow is 8–10days. The average expected survival time for circulating platelets is4–5 days, an average of the entire population. The average survival timefor platelets after isolation from the body is about 5 days at roomtemperature.

The current standard and approved method for platelet storage is in aplatelet bag at room temperature and limited to five days. The storagetime is presumably limited by a decrease in pH due to increased lactateassociated with anaerobic metabolic activity. Furthermore, the bag ofplatelets in plasma must be constantly in motion on a rocker to preventaggregation. One of the disadvantages associated with preservingplatelets under room temperature is the growth of bacteria in theplatelet suspension. Platelets in a suspension stored in a refrigerator,albeit with suppressed bacteria growth, tend to activate upon contactingeach other and aggregate.

Several approaches such as cryopreservation (freezing) techniques haveyielded an increased number of platelets following storage. However,there is a limitation in the functional capacity and persistence ofplatelets in circulation-that are recovered from such preservationconditions by using these methods. Freezing temperatures require the useof cryoprotectors such as DMSO (dimethyl sulfoxide) (Valeri, Feingold,and Marchionni, Blood, vol. 43, No. 1 (January)1974) and THROMBOSOJ toprevent damage to these biological materials. However, thesecryoprotectors are cytotoxic, and typically leave a significant portionof the platelets with either reduced or no functional ability. Moreover,cryoprotectors usually require time-consuming preparation, such asrinsing processes, before the materials can be used, and cryoprotectorresidues often still remain afterwards. Freezing processes can storeerythrocytes for more than 30 days, and leukocytes up to 12 hours only.

Other attempts to preserve platelets have included adding plateletsactivation inhibitors (Bode, Holme, Heaton and Swanson, Vox Sang, 60:105–112 (1991); U.S. Pat. No. 5,622,867) or gelatin into thepreservation medium (U.S. Pat. No. 2,786,014).

A need continues to exist for a storage system that will storebiological materials, particularly platelets, for an extended period oftime and still maintains their viability and bioactivity.

SUMMARY OF THE INVENTION

The present invention relates to compositions, methods and apparatusesfor the extended storage of biological material and, in particular,platelets.

According to one embodiment, a platelet composition suitable for directtransfusion into a patient is provided comprising: a preservation mediumcomprising plasma and a gel-forming material in a concentration relativeto the plasma such that the medium is in a sufficiently fluent state ata first temperature to allow platelets to move within the medium and isin a sufficiently gelatinous state at a second, lower temperature tosubstantially prevent platelets from moving freely within the medium;and platelets.

According to this embodiment, the first temperature is preferably about37° C. and the second temperature is preferably about 5° C.

According to another embodiment, a platelet composition suitable fordirect transfusion into a patient is provided comprising: a preservationmedium comprising plasma and a gel-forming material in a concentrationrelative to the plasma such that the medium is in a sufficiently fluentstate at a first temperature to allow platelets to move within themedium and is in a sufficiently gelatinous state at a second, lowertemperature to substantially prevent platelets from moving freely withinthe medium; and platelets which have been stored within the preservationmedium in a gelatinous state for at least 3 days where at least 50% ofthe platelets are intact and functional after the at least 3 days.

According to this embodiment, the first temperature is preferably about37° C. and the second temperature is preferably about 5° C.

Also according to this embodiment, the platelets may be stored withinthe preservation medium for at least 5 days, more preferably at least 7days. Also according to this embodiment, the platelets may be storedwithin the preservation medium for between 3 and 20 days, morepreferably between 5 and 20 days. Longer storage of platelets is alsopossible.

Also according to this embodiment, the platelets may be stored withinthe preservation medium at a temperature less than 10° C. and preferablybetween −10° C. and 10° C. In one variation, the platelets are stored ata temperature between 0° C. and 10° C. at 1ATM, more preferably at atemperature between 0° C. and 5° C. at 1ATM. In another variation, theplatelets are stored within the preservation medium at a temperaturebetween −10° C. and 0° C. at a pressure greater than 10ATM, morepreferably at a temperature between −8° C. and −2° C. at a pressuregreater than 10ATM.

According to another embodiment, a platelet composition suitable fordirect transfusion into a patient is provided comprising: a preservationmedium comprising plasma and a gel-forming material in a concentrationrelative to the plasma such that the medium is in a sufficiently fluentstate at a first temperature to allow platelets to move within themedium and is in a sufficiently gelatinous state at a second, lowertemperature to substantially prevent platelets from moving freely withinthe medium; and platelets which have been stored within the preservationmedium in a gelatinous state for at least 1 day at a pressure of atleast 10 ATM and a temperature below 0° C. where at least 50% of theplatelets are intact and functional after the at least 1 day.

According to this embodiment, the first temperature is preferably about37° C. and the second temperature is preferably about 5° C.

Also according to this embodiment, the platelets may be stored withinthe preservation medium at a pressure of at least 30ATM, more preferablyat least 70ATM, most preferably at least 200ATM.

According to this embodiment, the platelets may be stored within thepreservation medium for at least 3 days, more preferably at least 5 daysand most preferably at least 7 days. Also according to this embodiment,the platelets may be stored within the preservation medium for between 2and 20 days, more preferably between 3 and 20 days. Longer storage ofplatelets is also possible.

The present invention also relates to a variety of methods for storingplatelets for direct transfusion into a patient. In one embodiment, themethod comprises:

-   (1) forming a fluent platelet composition comprising platelets and a    preservation medium including plasma and a gel-forming material in a    concentration relative to the plasma such that the medium is in a    sufficiently fluent state at a first temperature to allow platelets    to move within the medium and is in a sufficiently gelatinous state    at a second, lower temperature to substantially prevent platelets    from moving freely within the medium;-   (2) cooling the fluent preservation medium to form a sufficiently    gelatinous state to substantially prevent free movement of the    platelets within the preservation medium; and-   (3) storing the platelets within the preservation medium in a    gelatinous state for at least 3 days where at least 50% of the    platelets are intact and functional after the at least 3 days.

According to this embodiment, the first temperature is preferably about37° C. and the second temperature is preferably about 5° C.

According to this embodiment, the platelets may be stored within thepreservation medium for at least 5 days, more preferably at least 7days. Also according to this embodiment, the platelets may be storedwithin the preservation medium for between 3 and 20 days, morepreferably between 5 and 20 days. Longer storage of platelets is alsopossible.

Also according to this embodiment, the platelets may be stored withinthe preservation medium at a temperature less than 10° C. and preferablybetween −10° C. and 10° C. In one variation, the platelets are stored ata temperature between 0° C. and 10° C. at 1ATM, more preferably at atemperature between 0° C. and 5° C. at 1ATM. In another variation, theplatelets are stored within the preservation medium at a temperaturebetween −10° C. and 0° C. at a pressure greater than 10ATM, morepreferably at a temperature between −8° C. and −2° C. at a pressuregreater than 10ATM.

According to another embodiment, a method is provided for storingplatelets for direct transfusion into a patient comprising:

-   (1) forming a fluent platelet composition comprising platelets and a    preservation medium including plasma and a gel-forming material in a    concentration relative to the plasma such that the medium is in a    sufficiently fluent state at a first temperature to allow platelets    to move within the medium and is in a sufficiently gelatinous state    at a second, lower temperature to substantially prevent platelets    from moving freely within the medium;-   (2) cooling the fluent preservation medium to form a sufficiently    gelatinous state to substantially prevent free movement of the    platelets within the preservation medium; and-   (3) storing the platelets within the preservation medium in a    gelatinous state for at least 1 day at a temperature below 0° C. and    at a pressure of at least 10ATM where at least 50% of the platelets    are intact and functional after the at least 1 day.

According to this embodiment, the first temperature is about 37° C. andthe second temperature is about 5° C.

According to this embodiment, the platelets are preferably stored withinthe preservation medium at a pressure of at least 30 ATM, morepreferably at a pressure of at least 70 ATM, most preferably at apressure of at least 200 ATM.

According to this embodiment, the platelets may be stored within thepreservation medium for at least 3 days, more preferably at least 5days, most preferably at least 7 days. Also according to thisembodiment, the platelets may be stored within the preservation mediumfor between 3 and 20 days, more preferably between 5 and 20 days. Longerstorage of platelets is also possible.

In regard to all of the above compositions and methods, it preferredthat at least 65% of the platelets are intact and functional afterstorage, more preferably at least 75% of the platelets, most preferablyat least 85% of the platelets.

Also in regard to all of the above compositions and methods, thegel-forming material preferably constitutes between 0.2% and 4% of thepreservation medium although the concentration may vary depending on theparticular gel-forming material used. Examples of gel-forming materialthat may be used include, but are not limited to gelatin, agarose, agar,pectin, carob cassia and natural or synthetic water soluble gum such asxanthan gum, konjac gum, guar gum, gum arabic, sodium alginate,carrageenan, irgacanth gum and hydroxyethyl methacrylaic.

Also in regard to all of the above compositions and methods, thepreservation medium may further include an energy source. The energysource preferably constitutes between 0 and 5% of the preservationmedium, more preferably between 0.25 and 5% of the preservation medium,and most preferably between 0.5 and 5% of the preservation medium. Awide variety of energy sources may be used. Most typically, the energysource is a carbohydrate, such as a sugar. Particular examples of energysources include glucose, sucrose, mannose, fructose and galactose.

Also in regard to all of the above compositions and methods, thepreservation medium may further include water-soluble salts. The saltpreferably constitutes between 0 and 2% of the preservation medium.Examples of salts include, but are not limited to sodium chloride,potassium chloride, magnesium chloride, sodium phosphate, potassiumphosphate and sodium gluconate.

Also in regard to all of the above compositions and methods, thepreservation medium may further include an anticoagulant. Examples ofanticoagulants that may be used include heparin, citrate dextrose,citrate phosphate dextrose, amantadine, ajoene and ticlopidine.

Also in regard to all of the above compositions and methods, thepreservation medium may further include amino acids. Examples of aminoacids that may be used include arginine, lysine, aspartate andglutamate.

The present invention also relates to an apparatus for preservingbiological materials. In one embodiment, the apparatus includes achamber having a mouth and a lip, the lip having an inside surface and atop surface, the inside surface and the top surface of the lip meetingat a first radius, the top surface of the lip having a channel. Theapparatus also includes a cover configured to mate with and seal thechamber, the cover having a bottom surface, the bottom surface having aprotrusion and a sealing structure, the bottom surface of the cover andthe protrusion meeting at a second radius, the protrusion being insertedinto the mouth of the chamber when the cover is mated with the chamber,the protrusion having a side surface, the side surface of the protrusionand the inside surface of the lip defining a first gap and beingsubstantially parallel when the cover is mated with the chamber, thebottom surface of the cover and the top surface of the lip defining asecond gap and being substantially parallel when the cover is mated withthe chamber, the second gap having a length greater than a width of thefirst gap, the sealing structure being inserted into the channel of thelip when the cover is mated with the chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of In K versus temperature for rate of biochemicalreaction.

FIG. 2 the phase transition lines for plasma and a 2.5% NaCl solution.

FIG. 3 shows another set of phase transition lines.

FIG. 4 shows a cross-sectional view of a biological materialpreservation apparatus of the present invention.

FIGS. 5A–5B shows a side cutaway and top views, respectively, of thechamber of the preservation apparatus.

FIG. 6 shows a side view of the cover of the preservation apparatus.

FIG. 7A–7C show a cover retaining device of the preservation apparatus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions, methods and apparatuses forstoring biological materials and, in particular, platelets, for anextended period of time. According to the present invention,functionally intact platelets can be recovered at high yields and useddirectly for platelet transfusions clinically.

1. Platelet Composition

The present invention provides various platelet compositions suitablefor direct transfusion into a patient. According to one embodiment, aplatelet composition comprises: a preservation medium comprising plasmaand a gel-forming material in a concentration relative to the plasmasuch that the medium is in a sufficiently fluent state at a firsttemperature to allow platelets to move within the medium and is in asufficiently gelatinous state at a second, lower temperature tosubstantially prevent platelets from moving freely within the medium;and platelets.

According to this embodiment, the first temperature is preferably about37° C. and the second temperature is preferably about 5° C.

Platelets tend to activate upon contacting each other and aggregate.When a gel-forming material is added into a suspension of platelets inplasma, the resulted platelet preservation medium is in a sufficientlyfluent state such that the platelets can move and distribute discretelywithin the medium following moderate agitations. When the plateletcomposition is cooled, the gel-forming material causes the preservationmedium to become sufficiently gelatinous so as to form a physicalbarrier between the already distributed platelets. In this regard,platelets are prevented from moving freely within the gelatinous medium.The gelatinous medium also provides a structural support that maintainsplatelet morphology and minimizes deformation of the platelet membranewhen the interior volume of the platelet changes during the coolingprocess. The gelatinous medium also lowers platelet metabolism bydecreasing biochemical exchanges between the platelet and itsenvironment. Inclusion of plasma in the preservation medium is believedto enhance platelet survival by simulating the platelet's nativeenvironment. Thus, retention of the functional integrity of platelets isimproved under the storage conditions provided by the present invention.As a result, a higher percentage of the platelets can still performtheir biological functions, such as promoting blood clotting, afterbeing stored according to present invention.

The shelf-life of platelets may be successfully extended by storing theplatelets in the preservation medium in a gelatinous state. Theplatelets may be stored within the preservation medium for at least 3days, more preferably at least 5 days, and most preferably at least 7days where at least 50% of the platelets are intact and functional afterthe storage period.

Also according to this embodiment, the platelets may be stored withinthe preservation medium for between 3 and 20 days, more preferablybetween 5 and 20 days. Longer storage of platelets is also possible.

Also according to this embodiment, the platelets may be stored withinthe preservation medium at a temperature less than 10° C. and preferablybetween −10° C. and 10° C. In one variation, the platelets are stored ata temperature between 0° C. and 10° C. at 1 ATM, more preferably at atemperature between 0° C. and 5° C. at 1 ATM. In another variation, theplatelets are stored within the preservation medium at a temperaturebetween −10° C. and 0° C. at a pressure greater than 10 ATM, morepreferably at a temperature between −8° C. and −2° C. at a pressuregreater than 10 ATM.

According to another embodiment, a platelet composition suitable fordirect transfusion into a patient is provided comprising: a preservationmedium comprising plasma and a gel-forming material in a concentrationrelative to the plasma such that the medium is in a sufficiently fluentstate at a first temperature to allow platelets to move within themedium and is in a sufficiently gelatinous state at a second, lowertemperature to substantially prevent platelets from moving freely withinthe medium; and platelets which have been stored within the preservationmedium in a gelatinous state for at least 1 day at a pressure of atleast 10 ATM and a temperature below 0° C. where at least 50% of theplatelets are intact and functional after the at least 1 day.

According to this embodiment, the first temperature is preferably about37° C. and the second temperature is preferably about 5° C.

According to this embodiment, the platelets may be stored within thepreservation medium at a pressure of at least 30ATM, more preferably atleast 70 ATM, most preferably at least 200 ATM.

According to this embodiment, the platelets may be stored within thepreservation medium for at least 3 days, more preferably at least 5days, most preferably at least 7 days. Also according to thisembodiment, the platelets may be stored within the preservation mediumfor between 3 and 20 days, more preferably between 5 and 20 days. Longerstorage of platelets is also possible.

In regard to all of the compositions of the present invention, itpreferred that at least 65% of the platelets are intact and functionalafter storage, more preferably at least 75% of the platelets, mostpreferably at least 85% of the platelets.

The gel-forming material preferably constitutes between 0.2% and 4% ofthe preservation medium although the concentration may vary depending onthe particular gel-forming material used. Examples of gel-formingmaterial that may be used include, but are not limited to, gelatin,agarose, agar, pectin, carob cassia and natural or synthetic watersoluble gums. Many of the gel-forming materials are commerciallyavailable. They are typically extracted from natural sources and areoften used as additive to various foods. Examples of water solublepolysaccharide gums include xanthan gum, konjac gum, guar gum, gumarabic, sodium alginate, carrageenan and irgacanth gum. Synthetic watersoluble gel-forming material includes hydroxyethyl methacrylaic.

The preservation medium may further include an energy source forincreasing hypertonicity of the medium. The energy source preferablyconstitutes between 0 and 5% of the preservation medium, more preferablybetween 0.25 and 5% of the preservation medium, and most preferablybetween 0.5 and 5% of the preservation medium.

A wide variety of energy sources may be used. Most typically, the energysource is a carbohydrate, such as a sugar. Particular examples of energysources include glucose, sucrose, mannose, fructose and galactose.Moreover, sucrose may repair damage in the cell membrane and glucoseprovides nutrients to sustain cell metabolism in the oxygen-poorconditions caused by the cooling process. Carbohydrates such as sucroseand glucose bind water, thus promoting gel formation and inhibitingosmotic pressure build-up within the platelets.

The preservation medium may further include water soluble salts. Thesalt preferably constitute between 0 and 2% of the preservation medium.Examples of salts include, but are not limited to, sodium chloride,potassium chloride, magnesium chloride, sodium phosphate, potassiumphosphate and sodium gluconate. For example, sodium chloride preventshemolysis by inhibiting the flow of water to the platelets duringcooling. As the platelets are cooled below 20° C., the cytoplasm changesfrom a colloid to a gel, and free water leaves the cell. As theplatelets are cooled even further, the hypertonic concentration of NaClprevents water from reentering the platelets. Sodium chloride alsolowers the freezing point of blood plasma by 2.5° C.

The preservation medium may further include an anticoagulant. Examplesof anticoagulants that may be used include heparin, citrate dextrose,citrate phosphate dectrose, amantadine, ajoene and ticlopidine.

The preservation medium may further include amino acids. Examples ofamino acids that may be used include arginine, lysine, aspartate andglutamate.

In a preferred embodiment, the preservation medium includes 1 to 3%gelatin. As it is cooled, the gelatin causes solidification of thepreservation medium and the resulted gel matrix forms a physical barrierbetween platelets. Thus, the gel matrix formed suspends cells in thepreservation medium and reduces sedimentation and clumping of platelets.

In a more preferred embodiment, the preservation solution includes 1.0to 3.0% gelatin, 1.0 to 2.0% glucose, 1.0 to 3.0% sucrose, and 0.2 to0.6% NaCl.

2. Methods for Platelet Storage

The present invention provides a variety of methods for storingplatelets for direct transfusion into a patient. According to oneembodiment, the method comprises the following steps:

1) forming a fluent platelet composition comprising platelets and apreservation medium including plasma and a gel-forming material in aconcentration relative to the plasma such that the medium is in asufficiently fluent state at a first temperature to allow platelets tomove within the medium and is in a sufficiently gelatinous state at asecond, lower temperature to substantially prevent platelets from movingfreely within the medium;

2) cooling the fluent preservation medium to form a sufficientlygelatinous state to substantially prevent free movement of the plateletswithin the preservation medium; and

3) storing the platelets within the preservation medium in a gelatinousstate for at least 3 days where at least 50% of the platelets are intactand functional after the at least 3 days.

According to this embodiment, the first temperature is preferably about37° C. and the second temperature is preferably about 5° C., althoughdifferent first and second temperatures may be employed.

For example, the platelet composition is formed by suspending plateletsin the preservation medium that includes a gel-forming material andplasma at about 37° C. The preservation medium is in a fluent state sothat the platelets can distribute discretely within the medium followingmoderate agitations. The platelet composition can be cooled gradually toabout 5° C. where the gel-forming material causes the preservationmedium to become sufficiently gelatinous so as to form a physicalbarrier between the already distributed platelets. Under suchconditions, platelets are prevented from moving freely within thegelatinous medium and activating upon contacting each other. As aresult, the platelets can be stored for a prolonged period of time andstill maintain their functional integrity.

According to this embodiment, the platelets may be stored within thepreservation medium for at least 3 days, more preferably at least 5 daysand most preferably at least 7 days. Also according to this embodiment,the platelets may be stored within the preservation medium for between 3and 20 days, more preferably between 5 and 20 days. Longer storage ofplatelets is also possible.

Also according to this embodiment, the platelets may be stored withinthe preservation medium at a temperature less than 10° C. and preferablybetween −10° C. and 10° C. In one variation, the platelets are stored ata temperature between 0° C. and 10° C. at 1 ATM, more preferably at atemperature between 0° C. and 5° C. at 1 ATM.

In another embodiment, a method is provided to store platelets atsubzero temperatures and under pressure higher than atmosphericpressure. The method comprises the following steps:

1) forming a fluent platelet composition comprising platelets and apreservation medium including plasma and a gel-forming material in aconcentration relative to the plasma such that the medium is in asufficiently fluent state at a first temperature to allow platelets tomove within the medium and is in a sufficiently gelatinous state at asecond, lower temperature to substantially prevent platelets from movingfreely within the medium;

2) cooling the fluent preservation medium to form a sufficientlygelatinous state to substantially prevent free movement of the plateletswithin the preservation medium; and

3) storing the platelets within the preservation medium in a gelatinousstate for at least 1 day at a temperature below 0° C. and at a pressureof at least 10 ATM where at least 50% of the platelets are intact andfunctional after the at least 1 day.

According to this embodiment, the platelets are preferably stored withinthe preservation medium at a pressure of at least 30 ATM, morepreferably at a pressure of at least 70 ATM, most preferably at apressure of at least 200 ATM.

In another variation, the platelets are stored within the preservationmedium at a temperature between −10° C. and 0° C. at a pressure greaterthan 10 ATM, more preferably at a temperature between −8° C. and −2° C.at a pressure greater than 10 ATM.

Also according to this embodiment, the platelets may be stored withinthe preservation medium for at least 3 days, more preferably at least 5days, most preferably at least 7 days. Also according to thisembodiment, the platelets may be stored within the preservation mediumfor between 3 and 20 days, more preferably between 5 and 20 days. Longerstorage of platelets is also possible.

By using these methods, platelets are stored under conditions wheretheir metabolism and biochemical reactions slow down and theirfunctional integrity is preserved. The platelet composition stored atlow temperature can be brought to a condition ready for transfusion intoa patient by warming the composition up to about 37° C. where thegel-like composition melts to a sufficiently fluent state.

3. Physics of Subzero Pressurized Storage

Temperature is one of the most important parameters to be consideredwhen storing living biological materials. When the temperature inside acell drops too low, irreversible biochemical and structural changesoccur. Several hundred biochemical reactions take place concurrently inthe living cell. The rate of these biochemical reactions depends onseveral factors, including pressure, temperature, viscosity of theenvironment, pH, and concentrations of reactive molecules.

A metabolic process typically includes a series of intermediateprocesses, in which a substrate S is converted into a series ofintermediate products X₁, X₂, X₃. . . before being converted into afinal product P. For each of these intermediate processes, the reactionsmay be catalyzed with different enzymes E₀, E₁, E₂. . . :E₀ E₁ E₂S→X₁→X₂→X₃ . . . →P  Equation (1)

Under normal conditions, the volume of substrate S transformed per unitof time equals the volume of product P obtained per unit of time:

$\begin{matrix}{{- \frac{\mathbb{d}\lbrack S\rbrack}{\mathbb{d}t}} = {+ \frac{\mathbb{d}\lbrack P\rbrack}{\mathbb{d}t}}} & {{Equation}\mspace{14mu}(2)}\end{matrix}$where [S] and [P] are the concentrations of substrate S and product P.The concentration of the intermediate products [X₁], [X₂], [X₃] undersuch conditions should also be constant:

$\begin{matrix}{\frac{\mathbb{d}\left\lbrack X_{1} \right\rbrack}{\mathbb{d}t} = {\frac{\mathbb{d}\left\lbrack X_{2} \right\rbrack}{\mathbb{d}t} = {\frac{\mathbb{d}\left\lbrack X_{3} \right\rbrack}{\mathbb{d}t} = 0}}} & {{Equation}\mspace{14mu}(3)}\end{matrix}$

Therefore, for each intermediate product; its rate of formation equalsits rate of transformation. The concentrations of each intermediateproduct may be expressed in terms of the rates of formation andtransformation:

$\begin{matrix}{\frac{\mathbb{d}\left\lbrack X_{3} \right\rbrack}{\mathbb{d}t} = {{- \frac{\mathbb{d}\left\lbrack X_{2} \right\rbrack}{\mathbb{d}t}} = {K_{2} \cdot \left\lbrack X_{2} \right\rbrack}}} & {{Equation}\mspace{14mu}(4)}\end{matrix}$where K₂ is the constant of rate reaction constant of transformation ofproduct X₂ and formation of product X₃. For steady state:

$\begin{matrix}{{- \frac{\mathbb{d}\lbrack S\rbrack}{\mathbb{d}t}} = {{+ \frac{\mathbb{d}\left\lbrack X_{1} \right\rbrack}{\mathbb{d}t}} = {{+ \frac{\mathbb{d}\left\lbrack X_{2} \right\rbrack}{\mathbb{d}t}} = {{{+ \frac{\mathbb{d}\left\lbrack X_{3} \right\rbrack}{\mathbb{d}t}}\ldots} = {+ \frac{\mathbb{d}\lbrack P\rbrack}{\mathbb{d}t}}}}}} & {{Equation}\mspace{14mu}(5)}\end{matrix}$From the above it follows that:K ₁ ●[X ₁ ]=K ₂ ●[X ₂][X ₁ ]:[X ₂ ]=K ₂ :K ₁  Equation (6)

Therefore, the concentration of each intermediate product is determinedby its rate constants of formation and transformation.

Temperature dependence is defined by constant K of the rate of chemicalreaction to Arrenius:K=A●_(e) ^(−E/RT)  Equation (7)

where

A is the constant coefficient in some temperature interval;

E is the activating energy of chemical reaction per 1 mol of thesubstance;

R is the universal gas constant; and

T is the absolute temperature.

For most biochemical reactions, E>>RT. Taking the natural logarithm ofboth sides of Equation (7) gives:

$\begin{matrix}{{\ln\mspace{14mu} K} = {\ln\mspace{14mu} A\frac{- E}{RT}}} & {{Equation}\mspace{14mu}(8)}\end{matrix}$

FIG. 1 shows a graph of 1n K versus temperature. From 30° C. to 37° C.,A is constant. For different chemical reactions E and A are different.As temperature decreases, there is a misbalance of reactions rates andEquation (5) no longer holds. This means the intermediate productconcentrations corresponding to each of the biochemical reactions beginto change. This begins breakdown of cell structures, including the cellmembrane, and can end in cell death.

Chemical reactions are either exothermic or endothermic, i.e. theyeither give off or absorb energy. Reactions taking place duringhydrolysis can release large amounts of energy. The oxidation of 1 molof glucose releases 2883 kJ of energy. Should the biochemical reactionrates slow down too much, irreversible process begin to take placefinally leading to total destruction of the cell. Therefore, coefficientA becomes a function of temperature T.

As the temperature drops below 20° C., the lipid bi-layer of the cellmembrane undergoes a phase transmission from a colloid to a gel. Theviscosity of a gel is much higher then that of its colloid.Consequently, rates of diffusion and active transportation of moleculesthrough the cell membrane decrease sharply, resulting in a slowing downof the rate of biochemical reactions in a cell. As a result of the phasetransformation of the cell membrane, the surface area of the lipidbi-lay surface and cell size reduce considerably due to the loss ofwater from the cell.

As the density of osmo-active substances increases, water moleculesreturn to the cell thereby increasing osmotic pressure. Membrane tensionreaches a critical point and may lose its barrier function. Membranedamage develops, resulting in morphological and structural changes, aswell as loss of the ability for active adaptation.

As the temperature drops below 8° C., the cell cytoplasm undergoes aphase transformation into a gel. At this temperature, there is a sharpdecrease in diffusion rate and active transportation of molecules, aswell as in biochemical reaction rates.

As the temperature falls below −3° C., water crystallization begins tooccur both inside and outside the cell. In the absence ofcryoprotectors, water crystallization outside the cell leads to celldehydration, decreased cell size, and increased concentrations of saltand other substances inside the cell. Water crystallization inside thecell results in structural cell membrane destruction.

FIG. 2 shows the phase transition lines for plasma and a 2.5% NaClsolution. At normal pressures, plasma freezes at −2.5° C. Plasmacontains various chemical which lower the freezing point by interferingwith the formation of the crystal lattice structure of ice. Cellstructures can be cooled to −4° C. to −3° C. without watercrystallization into cytoplasm. At normal pressures, the 2.5% NaClsolution freezes at −1.7° C.

FIG. 3 shows the phase transition lines for water and a 2.5% NaClsolution. The addition of NaCl to water as lowers the freezing point,and thus allows lower temperatures to be achieved for a given pressure.The line shows one example of how a biological material may be subjectedto a combination of high pressure and low temperature to preventfreezing.

4. Apparatus for Subzero Pressurized Storage

The present invention also provides apparatuses for the extended storageof biological materials, and, in particular, platelets. For example, theapparatuses can be used to store platelets suspended in a preservationmedium comprising plasma and a gel-forming material in a concentrationrelative to the plasma such that the medium is in a sufficiently fluentstate at about 37° C. to allow platelets to move within the medium andis in a sufficiently gelatinous state at about 5° C. to substantiallyprevent platelets from moving freely within the medium.

FIG. 4 shows an assembled view of one embodiment of a biologicalmaterial preservation apparatus 100 of the present invention.Preservation apparatus 100 includes a chamber 110 and a cover 130.

FIGS. 5A–5B show side cutaway and top views, respectively, of chamber110. Chamber 110 includes a mouth 111 and a lip 112. Lip 112 includes aninside surface 113 and a top surface 114. Inside surface 113 and topsurface 114 meet at a first radius r₁. Top surface 114 includes achannel 115. Channel 115 may have a sealing device 116 seated at abottom of channel 115, such as an O-ring or rubber gasket. Chamber 110may be manufactured in different sizes to accommodate a platelet bag,blood donation bag, heart, liver, kidney, or other bags and biologicalmaterials.

FIG. 6 shows a cutaway view of cover 130. Cover 130 is configured tomate with and seal chamber 110. Cover 130 includes a bottom surface 131.Bottom surface 131 includes a protrusion 132 and a sealing structure133. Bottom surface 131 and protrusion 132 meet at a second radius r₂.Protrusion 132 is inserted into mouth 111 of chamber 110 when cover 130is mated with chamber 110. Protrusion 132 includes a side surface 134.Side surface 134 of protrusion 132 and inside surface 113 of lip 112define a first gap 140 and are substantially parallel when cover 130 ismated with chamber 110. Bottom surface 132 of cover 130 and top surface118 of lip 114 define a second gap 141 and are substantially parallelwhen cover 130 is mated with chamber 110. Second gap 141 has a lengthgreater than a width of first gap 140. Sealing structure 133 is insertedinto channel 115 of lip 112 when cover 130 is mated with chamber 110.

Cover 130 may be made to be a spherical section, which allows cover 130to be made lighter and with less material than a flat cover 130 withoutsacrificing strength. When preservation apparatus 100 is filled with,for example, saline solution and then cooled below the freezing point,ice will begin to form along the walls of chamber 110 and cover 130. Icewill form in first gap 140 and second gap 141 and help to seal chamber110. Thus, the high pressures within chamber 110 are largely borne bythis ice seal, thus minimizing the need to make channel 115, sealingdevice 116, and sealing structure 133 extremely robust and capable ofwithstanding such high pressures. Channel 115, sealing device 116, andsealing structure 133 only need to withstand pressures of up to 10 atmbefore the ice seal takes over. The sizes of first gap 140 and second141 are not critical, but may be minimized so that ice fills them beforethe seal is subjected to pressures above 10 atm. In one embodiment,first gap 140 and second gap 141 may be less than 2.0 mm in width.Chamber 110 includes a suspension device 117 which prevents biologicalmaterial or bag placed within pressure chamber from coming into contactwith the walls of chamber 110. Suspension device 117 may be a net, aplatform, a spacer, or any other suitable device. Cover 130 may bedesigned to be sealed to chamber 110 directly, or with the aid of acover retaining device 135. Cover retaining device 135 may be designedto allow cover 130 to be installed and removed quickly and easily. Coverretaining device 135 may be coupled to chamber 110 via a bayonet-styleconnection, threads, or any other suitable coupling method. Coverretaining device 135 may include a centering pin 136 to keep coverretaining device 135 centered or attached to cover 130. Cover retainingdevice 135 may also include holes 137 to allow a wrench or other tool tobe used with cover retaining device 135. Cover retaining device 135 maybe produced in two separated pieces to simplify manufacturing. FIGS.7A–7C show cutaway and top views of a two-piece cover retaining device135.

Preservation apparatus 100 may include a pressure gauge 150 with anelastic membrane 151 placed within chamber 110. Pressure gauge 150 mayinclude a relief valve 152 which prevents pressure within preservationapparatus 100 from exceeding a predetermined maximum.

By utilizing the features of the present invention, the followingobjectives for preserving biological materials, in particular,platelets, are achieved:

1. Mechanically suspending the biological materials in a preservationmedium.

2. Storing the biological materials at the lowest possible temperaturewhile maintaining them in a liquid state. Under these conditions, therate of biochemical reactions are relatively slow and therefore, therates of change in the concentrations of intermediate products is small.

3. Slowly cooling solutions with platelets to allow free and safe waterflow from the cell to prevent membrane tension from reaching a burstingpoint during the phase transmission from a colloid to a gel. On theother hand, the cooling rate should be high enough to preventintermediate biochemical reactions from causing irreversible changes incell structure.

EXAMPLES

1. Method of Platelet Preservation

The following is one example of the method of the present invention forpreserving blood platelets. Heparin may be used as an anticoagulantbefore this process is begun.

(1) Mix the platelets with a preservation solution of 2.9% gelatin,0.44% sucrose, 1.17% glucose, and 0.49% NaCl.

(2) Seal the platelets and preservation solution into a storage bag,making sure that any air has been pumped out. The storage may be anystandard platelet storage bag such as a flexible silicone rubber bag.

(3) Cool the platelets and preservation solution to 15° C. within 1hour. Continuous agitation is required until the preservation solutionbecomes a gel.

(4) Cool the storage bag to 6° C. to 8° C. within 1 to 1.5 hours.

(5) Cool the preservation apparatus to 6° C. to 8° C.

(6) Insert the storage bag into the preservation apparatus using thesuspension device.

(7) Fill the preservation apparatus with a pressure transfer fluid of2.5% NaCl solution.

(8) Seal the preservation apparatus, making sure it is completely fulland no air is trapped inside.

(9) Cool the preservation apparatus to −7.5 0.2° C. within 1.5 to 2hours. The pressure transfer fluid is a fluid which expands when cooledor frozen, and thus will be able to exert a pressure upon the bag withinthe substantially fixed volume of the preservation apparatus. With the2.5% NaCl solution, the water will begin to freeze at the walls of thepressure chamber. As the ice is formed at the walls of the pressurechamber, the expansion will create the high pressures required withinthe preservation apparatus, which will be transferred by the unfrozenfluid immediately surrounding the storage bag to the storage bag. TheNaCl lowers the freezing point of the pressure transfer fluid, thusallowing the low temperatures required to be achieved before the entirevolume of the pressure transfer fluid becomes frozen. The preservationsolution has a lower freezing point than the pressure transfer fluid.The pressure inside the preservation apparatus will rise to 500 atm. Asice begins to form, pressure within the preservation apparatus willincrease because ice and water are essentially non-compressible. Therelationship between temperature and pressure here is consistent andpredictable. The combination of the preservation solution, the highstorage pressure, and the low storage temperature allows the plateletsto be stored for up to 15 days. Erythrocytes may be stored up to 30 daysand leukocytes up to 22 days using this method.

(10) When the platelets are needed for use, allow the preservationapparatus to thaw completely at room temperature, approximately 20° C.,before opening the preservation apparatus. Because the components in thepreservation solution are all nontoxic, the platelets may be usedimmediately without further preparation.

2. Studies of Platelet Survival

Human blood platelets suspended in plasma and contained in standardplatelet bags were mixed with a concentrated gelatin stock solution at37° C. The concentrated gelatin stock solution also contained sugar andsodium chloride. Typically, the amount of gelatin solution added was ¼the volume of plasma. For example, 25 ml of a gelatin stock solutioncontaining 3.5% gelatin and 10% glucose was added to 100 ml of plasmawith platelets, resulting in a final solution at 0.7% gelatin and 2%glucose. The final concentration of platelets in the platelet bag isabout 300,000 per μl.

The platelet compositions were stored at a refrigerator temperature orin an preservation apparatus according to the present invention atsubzero temperatures. Following certain periods of time (n days)storage, the platelet compositions were warmed to about 37° C. andanalyzed for post-storage (Dn) activity, such as platelet aggregation,as compared to the activity of platelets before storage (D0) by usingstandard methods performed by hospital clinical laboratories.

Typically, platelet aggregation is performed by adding a stimulus, suchas 10 μM adenosine diphosphate (ADP) and 14 μg ristocetin, to asuspension of platelets in a curvette or on a slide. Methods and amountsof stimuli typically used are well known to those skilled in the art.The stimulating agent binds to receptors on the platelets and causes theplatelets to release substances from granules and initiates a cascade ofevents resulting in platelets binding to each other and falling out ofthe suspension. Typically, the aggregation of platelets is indicated byan increased ability of the solution to allow passage of light(increased % transmission or decreased turbidity). The time plateletsrespond to each stimulus was recorded in seconds. The survival rate ofplatelets was measured by counting post-storage platelets with intactmorphology under a microscope and comparing with the platelets beforethe storage.

Table I lists the constituents of the preservation medium, the survivalrates of the platelets and aggregation response times of the plateletswhen exposed to ristocetin or ADP and after the platelets have beenstored in preservation media for a listed period of time (n days) at arefrigerator temperature 4° C.

TABLE I Aggregation Response (sec.) n days Sur- Ristocetin ADP GelatinSucrose Glucose NaCl at 4° C. vival D_(o)/D_(n) D_(o)/D_(n) 0.45% 2.0%2.0% 0.5% 5 d  82% 9/7 9/8  0.7% 2.0% 2.0% 0.5% 5 d 100% 14/7  11/7  1.5% 2.0% 2.0% 0.5% 5 d  88% 14/12 17/12Table II lists the constituents of the preservation medium, the survivalrates of the platelets and aggregation response times of the plateletswhen exposed to ristocetin and adenosine diphosphate (ADP) after theplatelets have been stored in preservation media for a listed period oftime at a sub-zero temperature (−4 to −10° C.).

TABLE II Aggregation n days Response (sec.) at Sur- Ristocetin ADPGelatin Sucrose Glucose NaCl <0° C. vival D_(o)/D_(n) D_(o)/D_(n)  5 d 6% 11/23 11/43 0.5% 0.5% 1.0% 0.5%  5 d  76% 9/9  9/10 1.5% 2.0% 1.0%0.5%  5 d 100% 11/8  10/9  1.5% 1.5% 1.0% 0.5%  5 d 100%  9/14 10/170.7% 2.0% 2.0% 0.5% 11 d  45%  9/10 10/13 1.5% 2.0% 2.0% 0.5% 11 d >50%9/8 11/8  0.7% 2.0% 2.0% 0.5% 13 d  86% 9/4 10/7  1.5% 2.0% 2.0% 0.5% 13d 100% 11/6  14/5 

As can be seen from the results shown in Table I and II, platelets werestored for five or more days with high survival rates.

The foregoing description of the invention has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise forms disclosed.Many modifications and variations will be apparent. It is intended thatthe scope of the invention be defined by the following claims and theirequivalents.

1. A platelet composition suitable for direct transfusion into a patientcomprising: a preservation medium comprising plasma, an anti-coagulant,and a gel-forming material in a concentration relative to the plasmasuch that the medium is in a sufficiently fluent state at about 37° C.to allow platelets to move within the medium and is in a sufficientlygelatinous state at about 5° C. to substantially prevent platelets frommoving freely within the medium; and platelets which have been stored ina non-frozen state within the preservation medium in a gelatinous statefor at least 3 days at a pressure of at least 1 ATM and a temperaturebelow 10° C. where at least 50% of the platelets are intact andfunctional based on adenosine diphosphate induced platelet aggregationassay after at least 3 days.
 2. The platelet composition according toclaim 1, wherein the platelets are stored within the preservation mediumfor at least 5 days where at least 50% of the platelets are intact andfunctional based on adenosine diphosphate induced platelet aggregationassay after at least 5 days.
 3. The platelet composition according toclaim 1, wherein the platelets are stored within the preservation mediumfor at least 7 days where at least 50% of the platelets are intact andfunctional based on adenosine diphosphate induced platelet aggregationassay after at least 7 days.
 4. The platelet composition according toclaim 1, wherein the platelets are stored within the preservation mediumfor between 3 and 20 days where at least 50% of the platelets are intactand functional based on adenosine diphosphate induced plateletaggregation assay after at least 3 and 20 days.
 5. The plateletcomposition according to claim 1, where the platelets are stored for atleast 3 days within the preservation medium at a temperature less than10° C.
 6. The platelet composition according to claim 1, where theplatelets are stored for at least 3 days within the preservation mediumat a temperature less than 5° C.
 7. The platelet composition accordingto claim 1, where the platelets are stored for at least 3 days withinthe preservation medium at a temperature between −10° C. and 10° C. 8.The platelet composition according to claim 1, where the platelets arestored for at least 3 days within the preservation medium at atemperature between 0° C. and 10° C. at 1 ATM.
 9. The plateletcomposition according to claim 1, where the platelets are stored for atleast 3 days within the preservation medium at a temperature between 0°C. and 5° C. at 1 ATM.
 10. The platelet composition according to claim1, where the platelets are stored for at least 3 days within thepreservation medium at a temperature between −10° C. and 0° C. at apressure greater than 10 ATM.
 11. The platelet composition according toclaim 1, where the platelets are stored for at least 3 days within thepreservation medium at a temperature between −8° C. and −2° C. at apressure greater than 10 ATM.
 12. The platelet composition according toclaim 1, where at least 65% of the platelets are intact and functionalbased on adenosine diphosphate induced platelet aggregation assay afterat least 3 days.
 13. The platelet composition according to claim 1,where at least 75% of the platelets are intact and functional based onadenosine diphosphate induced platelet aggregation assay after at least3 days.
 14. The platelet composition according to claim 1, where atleast 85% of the platelets are intact and functional based on adenosinediphosphate induced platelet aggregation assay after at least 3 days.15. The platelet composition according to claim 1, wherein thegel-forming material constitutes between 0.2% and 4% of the preservationmedium.
 16. The platelet composition according to claim 1, wherein thegel-forming material is selected from the group consisting of gelatin,agarose, agar, pectin, carob cassia, xanthan gum, konjac gum, guar gum,gum arabic, sodium alginate, carrageenan, irgacanth gum and hydroxyethylmethacrylaic.
 17. The platelet composition according to claim 1, whereinthe preservation medium further includes an energy source.
 18. Theplatelet composition according to claim 17, wherein the energy sourceconstitutes between 0 and 5% of the preservation medium.
 19. Theplatelet composition according to claim 17, wherein the energy sourceincludes a carbohydrate.
 20. The platelet composition according to claim17, wherein the energy source includes a sugar selected from the groupconsisting of glucose, sucrose, mannose, fructose and galactose.
 21. Theplatelet composition according to claim 1, wherein the anticoagulant isselected from the group consisting of heparin, citrate dextrose, citratephosphate dextrose, amantadine, ajoene and ticlopidine.
 22. A plateletcomposition suitable for direct transfusion into a patient comprising: apreservation medium comprising plasma, an anti-coagulant, and agel-forming material in a concentration relative to the plasma such thatthe medium is in a sufficiently fluent state at about 37° C. to allowplatelets to move within the medium and is in a sufficiently gelatinousstate at about 5° C. to substantially prevent platelets from movingfreely within the medium; and platelets.
 23. A platelet compositionsuitable for direct transfusion into a patient compnsing: a preservationmedium comprising plasma, an anti-coagulant, and a gel-forming materialin a concentration relative to the plasma such that the medium is in asufficiently fluent state at about 37° C. to allow platelets to movewithin the medium and is in a sufficiently gelatinous state at about 5°C. to substantially prevent platelets from moving freely within themedium; and platelets stored within the preservation medium for at least1 day at a temperature below 0° C. and at a pressure of at least 10 ATMwhere at least 50% of the platelets are intact and functional based onadenosine diphosphate induced platelet aggregation assay after at least1 day.
 24. The platelet composition according to claim 23, wherein theplatelets are stored within the preservation medium at a pressure of atleast 70 ATM.
 25. The platelet composition according to claim 23,wherein the platelets are stored within the preservation medium at apressure of at least 200 ATM.
 26. The platelet composition according toclaim 23, wherein the platelets are stored within the preservationmedium for at least 3 days where at least 50% of the platelets areintact and functional based on adenosine diphosphate induced plateletaggregation assay after at least 3 days.
 27. The platelet compositionaccording to claim 23, wherein the platelets are stored within thepreservation medium for at least 5 days where at least 50% of theplatelets are intact and functional based on adenosine diphosphateinduced platelet aggregation assay after at least 5 days.
 28. Theplatelet composition according to claim 23, wherein the platelets arestored within the preservation medium for between 3 and 20 days where atleast 50% of the platelets are intact and functional based on adenosinediphosphate induced platelet aggregation assay after at least 3 and 20days.
 29. The platelet composition according to claim 23, where at least65% of the platelets are intact and functional based on adenosinediphosphate induced platelet aggregation assay after at least 3 days.30. The platelet composition according to claim 23, where at least 75%of the platelets are intact and functional based on adenosinediphosphate induced platelet aggregation assay after at least 3 days.31. The platelet composition according to claim 23, where at least 85%of the platelets are intact and functional based on adenosinediphosphate induced platelet aggregation assay after at least 3 days.32. The platelet composition according to claim 23, wherein thegel-forming material constitutes between 0.2% and 4% of the preservationmedium.
 33. The platelet composition according to claim 23, wherein thegel-forming material is selected from the group consisting of gelatin,agarose, agar, pectin, carob cassia, xanthan gum, konjac gum, guar gum,gum arabic, sodium alginate, carrageenan, irgacanth gum and hydroxyethylmethacrylaic.
 34. The platelet composition according to claim 23,wherein the preservation medium further includes an energy source. 35.The platelet composition according to claim 34, wherein the energysource constitutes between 0 and 5% of the preservation medium.
 36. Theplatelet composition according to claim 34, wherein the energy sourceincludes a carbohydrate.
 37. The platelet composition according to claim34, wherein the energy source includes a sugar selected from the groupconsisting of glucose, sucrose, mannose, fructose and galactose.
 38. Theplatelet composition according to claim 22 or 23, wherein theanticoagulant is selected from the group consisting of heparin, citratedextrose, citrate phosphate dextrose, amantadine, ajoene andticlopidine.